Deregulation of cell cycle leads to cell transformation and cancer development. Here we present profiling the proteome dynamics using 2-DE and constructing the associated functional networks during the cell cycle of human hepatoma cells, Mahlavu. The protein dynamics was validated by hierarchical clustering analysis on the proteome, and by Northern blot assays on the selected 14-3-3 proteins. Of the 2665 protein spots, 201 with variation coefficient of expression dynamics >20% throughout the cell cycle were subjected to analysis. Degree of the global protein dynamics was phase dependent with the greatest in transitional phases of S/G2, G2/M, and G1/S. Concurrence of pathways coordinating cell-cycle progression versus arrest, and/or pathways regulating apoptosis versus antiapoptosis was always identified during the cell cycle, suggesting the existence of counteracting mechanisms for intracellular homeostasis. Data mining of the results suggested that the key transcription factors in G0/G1, G1/S, S, and G2/M were p53 and SP1, c-Myc, c-Myc and p53, and YY1 and c-Jun, respectively. Our findings for the first time provide insights into the regulation of mammalian cell-cycle progression at the proteome level, and grant a model to study disease mechanisms and to discover therapeutic targets for anticancer therapy.